The effects of glucose on aliphatic glucosinolate biosynthesis in Arabidopsis thaliana were investigated in this study by using mutants related to aliphatic glucosinolate biosynthesis and regulation, as well as glucose signalling. The results showed that glucose significantly increased the contents of individual and total aliphatic glucosinolates. Expression of MYB28 and MYB29, two key transcription factors in aliphatic glucosinolate biosynthesis, was also induced by glucose. Consistently, the increased accumulation of aliphatic glucosinolates and the up-regulated expression of CYP79F1 and CYP79F2 induced by glucose disappeared in the double mutant myb28myb29. MYB28 and MYB29 synergistically functioned in the glucose-induced biosynthesis of aliphatic glucosinolates, but MYB28 was predominant over MYB29. Interestingly, the content of total aliphatic glucosinolates and the expression level of MYB28 and MYB29 were substantially reduced in the glucose insensitive mutant gin2-1 and the ABA insensitive 5 (abi5-7) mutant compared with the wild type. In addition, total aliphatic glucosinolates accumulated much less in another sugar-insensitive RGS1 (regulator of G-protein signaling 1) mutant (rgs1-2) than in the wild type. These results suggest that glucose-promoted aliphatic glucosinolate biosynthesis is regulated by HXK1- and/or RGS1-mediated signalling via transcription factors, MYB28, MYB29, and ABI5.